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United States Patent |
5,618,830
|
Selnick
,   et al.
|
April 8, 1997
|
Dioxobutanoic acid derivatives as inhibitors of influenza endonuclease
Abstract
Dioxobutanoic acids substituted with piperidine or similar N-substituted
saturated cycloalkyls are found to inhibit the cap-dependent endonuclease
of influenza virus. These compounds are useful in the prevention or
treatment of infection by influenza virus and the treatment of influenza,
either as compounds, pharmaceutically acceptable salts, pharmaceutical
composition ingredients, whether or not in combination with other
antivirals, immunomodulators, antibiotics or vaccines. Methods of treating
influenza and methods of preventing or treating infection by influenza
virus are also described.
Inventors:
|
Selnick; Harold G. (Ambler, PA);
Baldwin; John J. (Gwynedd Valley, PA);
Ponticello; Gerald S. (Lansdale, PA);
Tomassini; Joanne E. (Lansdale, PA)
|
Assignee:
|
Merck & Co., Inc. (Rahway, NJ)
|
Appl. No.:
|
536294 |
Filed:
|
September 29, 1995 |
Current U.S. Class: |
514/358; 514/317; 514/318 |
Intern'l Class: |
A61K 031/44; A61K 031/445 |
Field of Search: |
514/358,317,318
|
References Cited
Attorney, Agent or Firm: Ayler; Sylvia A., Meredith; Roy D., Daniel; Mark R.
Parent Case Text
This is a division of application Ser. No. 08/324,190 filed Oct. 17, 1994,
now U.S. Pat. No. 5,475,109.
Claims
What is claimed is:
1. Pharmaceutical composition, for use in the treatment of influenza, in
the prevention of infection by influenza virus, or in the inhibition of
influenza cap-dependent endonuclease, comprising a pharmaceutically
acceptable carrier and an effective amount of a compound of the formula
##STR75##
or pharmaceutically acceptable salt, hydrate or crystal form thereof,
wherein:
X is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
Z is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
Y is --CH.sub.2 --, CO, SO.sub.2 --, or a bond;
R.sub.1 and R.sub.2 are independently selected from the following:
branched or unbranched C.sub.1-6 alkyl-, C.sub.1-6 alkyloxy-,
--N--C.sub.1-6 alkyl-, C.sub.3-8 cycloalkyl-, phenyl, naphthyl, pyridyl,
furanyl, thienyl, or quinolinyl, any of which may be substituted once or
twice with C.sub.1-5 alkyl, C.sub.3-8 cycloalkyl, phenyl, quinolinyl,
pyridyl, furanyl, thienyl, C.sub.1-6 -alkoxy, Br, F, or Cl.
2. Pharmaceutical composition, for use in the treatment of influenza, in
the prevention of infection by influenza virus, or in the inhibition of
influenza cap-dependent endonuclease, comprising a pharmaceutically
acceptable carrier and an effective amount of a compound of the formula
##STR76##
or pharmaceutically acceptable salt, hydrate or crystal form thereof,
wherein:
X is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
Z is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
R.sub.1 is
(i) phenyl, unsubstituted or substituted with halo; or
(ii) cyclohexyl;
R.sub.2 is
(i) H or
(ii) benzyl, unsubstituted or substituted with halo.
3. Pharmaceutical composition, for use in the treatment of influenza, in
the prevention of infection by influenza virus, or in the inhibition of
influenza cap-dependent endonuclease, comprising a pharmaceutically
acceptable carrier and an effective amount of a compound of the structure
##STR77##
named 4-[N-benzyl-3-(4-chlorobenzyl)piperidin-3-yl]2,4-dioxobutanoic acid
hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
4. Pharmaceutical composition, for use in the treatment of influenza, in
the prevention of infection by influenza virus, or in the inhibition of
influenza cap-dependent endonuclease, comprising a pharmaceutically
acceptable carrier and an effective amount of a compound of the structure
##STR78##
named 4-[N-benzyl-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic acid
hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
5. Pharmaceutical composition, for use in the treatment of influenza, in
the prevention of infection by influenza, or in the inhibition of
influenza cap-dependent endonuclease, comprising a pharmaceutically
acceptable carrier and an effective amount of a compound of the structure
##STR79##
named
4-N-(p-chlorobenzyl)-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic
acid hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
6. Pharmaceutical composition, for use in the treatment of influenza, in
the prevention of infection by influenza virus, or in the inhibition of
influenza cap-dependent endonuclease, comprising a pharmaceutically
acceptable carrier and an effective amount of a compound of the structure
##STR80##
named 4-[1-cyclohexylmethyl-4-(p-chlorobenzyl)piperidin-4-yl
]2,4-dioxobutanoic acid hydrochloride, or pharmaceutically acceptable
salt, hydrate or ester thereof.
7. A method of heating influenza, comprising administering to a mammal in
need of such treatment, an effective amount of a compound of the formula
##STR81##
or pharmaceutically acceptable salt, hydrate or crystal form thereof,
wherein:
X is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
Z is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
Y is --CH.sub.2 --, CO, SO.sub.2 --, or a bond;
R.sub.1 and R.sub.2 are independently selected from the following:
branched or unbranched C.sub.1-6 alkyl-, C.sub.1-6 alkyloxy-,
--N--C.sub.1-6 alkyl-, C.sub.3-8 cycloalkyl-, phenyl, naphthyl, pyridyl,
furanyl, thienyl, or quinolinyl, any of which may be substituted once or
twice with C.sub.1-5 alkyl, C.sub.3-8 cycloalkyl, phenyl, quinolinyl,
pyridyl, furanyl, thienyl, C.sub.1-6 -alkoxy, Br, F, or Cl.
8. A method of treating influenza, comprising administering to a mammal in
need of such treatment, an effective amount of a compound of the formula
##STR82##
or pharmaceutically acceptable salt, hydrate or crystal form thereof,
wherein:
X is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
Z is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
R.sub.1 is
(i) phenyl, unsubstituted or substituted with halo; or
(ii) cyclohexyl;
R.sub.2 is
(i) H or
(ii) benzyl, unsubstituted or substituted with halo.
9. A method of treating influenza, comprising administering to a mammal in
need of such treatment, an effective amount of a compound of the structure
##STR83##
named 4-[N-benzyl-3-(4-chlorobenzyl)piperidin-3-yl]2,4-dioxobutanoic acid
hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
10. A method of treating influenza, comprising administering to a mammal in
need of such treatment, an effective amount of a compound of the structure
##STR84##
named 4-[N-benzyl-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic acid
hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
11. A method of treating influenza, comprising administering to a mammal in
need of such treatment, an effective amount of a compound of the structure
##STR85##
named
4-N-(p-chlorobenzyl)-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic
acid hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
12. A method of treating influenza, comprising administering to a mammal in
need of such treatment, an effective amount of a compound of the structure
##STR86##
named
4-[1-cyclohexylmethyl-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic
acid hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
13. A method of preventing infection by influenza virus, comprising
administering to a mammal in need of such treatment, an effective amount
of the formula
##STR87##
or pharmaceutically acceptable salt, hydrate or crystal form thereof,
wherein:
X is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
Z is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
Y is --CH.sub.2 --, CO, SO.sub.2 --, or a bond;
R.sub.1 and R.sub.2 are independently selected from the following:
branched or unbranched C.sub.1-6 alkyl-, C.sub.1-6 alkyloxy-,
--N--C.sub.1-6 alkyl-, C.sub.3-8 cycloalkyl-, phenyl, naphthyl, pyridyl,
furanyl, thienyl, or quinolinyl, any of which may be substituted once or
twice with C.sub.1-5 alkyl, C.sub.3-8 cycloalkyl, phenyl, quinolinyl,
pyridyl, furanyl, thienyl, C.sub.1-6 -alkoxy, Br, F, or Cl.
14. A method of preventing infection by influenza virus, comprising
administering to a mammal in need of such treatment, an effective amount
of the formula
##STR88##
or pharmaceutically acceptable salt, hydrate or crystal form thereof,
wherein:
X is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
Z is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
R.sub.1 is
(i) phenyl, unsubstituted or substituted with halo; or
(ii) cyclohexyl;
R.sub.2 is
(i) H or
(ii) benzyl, unsubstituted or substituted with halo.
15. A method of preventing infection by influenza virus, comprising
administering to a mammal in need of such treatment, an effective amount
of a compound of the structure
##STR89##
named 4-[N-benzyl-3-(4-chlorobenzyl)piperidin-3-yl]2,4-dioxobutanoic acid
hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
16. A method of preventing infection by influenza virus, comprising
administering to a mammal in need of such treatment, an effective amount
of a compound of the structure
##STR90##
named 4-[N-benzyl-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic acid
hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
17. A method of preventing infection by influenza virus, comprising
administering to a mammal in need of such treatment, an effective amount
of a compound of the structure
##STR91##
named
4-N-(p-chlorobenzyl)-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic
acid hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
18. A method of preventing infection by influenza virus, comprising
administering to a mammal in need of such treatment, an effective amount
of a compound of the structure
##STR92##
named
4-[1-cyclohexylmethyl-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic
acid hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
19. A method of inhibiting influenza virus cap-dependent endonuclease,
comprising administering to a mammal in need of such treatment, an
effective amount of a compound of the formula
##STR93##
pharmaceutically acceptable salt, hydrate or crystal form thereof,
wherein:
X is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
Z is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
Y is --CH.sub.2 --, CO, SO.sub.2 --, or a bond;
R.sub.1 and R.sub.2 are independently selected from the following:
branched or unbranched C.sub.1-6 alkyl-, C.sub.1-6 alkyloxy-,
--N--C.sub.1-6 alkyl-, C.sub.3-8 cycloalkyl-, phenyl, naphthyl, pyridyl,
furanyl, thienyl, or quinolinyl, any of which may be substituted once or
twice with C.sub.1-5 alkyl, C.sub.3-8 cycloalkyl, phenyl, quinolinyl,
pyridyl, furanyl, thienyl, C.sub.1-6 -alkoxy, Br, F, or Cl.
20. A method of inhibiting influenza virus cap-dependent endonuclease,
comprising administering to a mammal in need of such treatment, an
effective amount of a compound of the structure
##STR94##
or pharmaceutically acceptable salt, hydrate or crystal form thereof,
wherein:
X is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
Z is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
R.sub.1 is
(i) phenyl, unsubstituted or substituted with halo; or
(ii) cyclohexyl;
R.sub.2 is
(i) H or
(ii) benzyl, unsubstituted or substituted with halo.
21. A method of inhibiting influenza virus cap-dependent endonuclease,
comprising administering to a mammal in need of such treatment, an
effective amount of a compound of the structure
##STR95##
named 4-[N-benzyl-3-(4-chlorobenzyl)piperidin-3-yl]2,4-dioxobutanoic acid
hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
22. A method of inhibiting influenza virus cap-dependent endonuclease,
comprising administering to a mammal in need of such treatment, an
effective amount of a compound of the structure
##STR96##
named 4-[N-benzyl-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic acid
hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
23. A method of inhibiting influenza virus cap-dependent endonuclease,
comprising administering to a mammal in need of such treatment an
effective amount of a compound of the structure
##STR97##
named
4-N-(p-chlorobenzyl)-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic
acid hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
24. A method of inhibiting influenza virus cap-dependent endonuclease,
comprising administering to a mammal in need of such treatment, an
effective amount of a compound of the structure
##STR98##
named
4-[1-cyclohexyhnethyl-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic
acid hydrochloride, or pharmaceutically acceptable salt, hydrate or ester
thereof.
Description
The present invention is concerned with compounds which inhibit an
endonuclease encoded by influenza virus. The compounds, or
pharmaceutically acceptable salts thereof, are of value in the prevention
of infection by influenza virus, and the treatment of infection by
influenza virus.
The present invention also relates to pharmaceutical compositions
containing the compounds and to a method of use of the present compounds
and other agents for the treatment of influenza and the viral infection by
influenza virus.
BACKGROUND OF THE INVENTION
A myxovirus designated influenza is the etiological agent of the common
"flu", an acute highly contagious viral disease characterized by sudden
onset, fever, prostration, and progressive intimation of the respiratory
mucous membrane.
The compounds of the present invention contain dioxobutanoic acids
substituted with piperidine and similar N-substituted saturated
cycloalkyls. Applicants demonstrate that the compounds of this invention
are inhibitors of the cap-dependent endonuclease of influenza virus. The
compounds were shown to be selective for influenza transcription by
testing in several related enzyme assays. The compounds were not
inhibitory in other polymerase assays including VSV transcription, HIV
reverse transcriptase, T7 phage, Hela cell RNA polymerase II and Hela cell
DNA polymerase .alpha. when tested at concentrations 100-500 fold above
the IC.sub.50 for influenza transcription; thereby showing specific
inhibition of influenza transcription. The inhibitory activity was
specific to cap-dependent influenza transcription and had no effect upon
influenza transcription primed cap-independently with the dinucleotide
ApG. The mode of action of the inhibitors was demonstrated to be
inhibition of cap-dependent endonuclease activity in an influenza cleavage
assay in which the dioxobutanoic acids had IC.sub.50 s similar to those
obtained in influenza transcription. Additionally, the inhibitor had no
effect upon transcription when primed with capped substrates which did not
undergo endonucleolytic processing, further confining inhibition of the
influenza endonuclease. The specificity of influenza cleavage inhibition
was demonstrated in nuclease counterscreens in which the compound had no
effect upon RNases A, T1, U1 and HIV RNase H when tested up to 100-fold
above the IC.sub.50 obtained in influenza cleavage.
BRIEF DESCRIPTION OF THE INVENTION
Compounds of Formula I, as herein defined, are disclosed. These compounds
are useful in the inhibition of influenza virus cap-dependent
endonuclease, the prevention of infection by influenza virus, and the
treatment of infection by influenza virus, either as compounds,
pharmaceutically acceptable salts, hydrates or esters, pharmaceutical
composition ingredients, whether or not in combination with other
antivirals, immunomodulators, antibiotics or vaccines. Methods of treating
influenza, methods of preventing infection by influenza virus, and methods
of treating infection by influenza virus are also disclosed.
______________________________________
ABBREVIATIONS
______________________________________
Activating Agent
HBT (HOBT or HOBt)
1-hydroxybenzotriazole hydrate
Condensing Agent
EDC 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide
______________________________________
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
This invention is concerned with the compounds of Formula I, combinations
thereof, or pharmaceutically acceptable salts thereof, in the inhibition
of influenza virus transcriptase, the prevention of infection by influenza
virus, and the treatment of infection by influenza virus. Compounds of
Formula I are defined as follows:
##STR1##
or pharmaceutically acceptable salt, hydrate or crystal form thereof,
wherein:
X is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
Z is --CH.sub.2 --, CH.sub.2 --CH.sub.2 --, or a bond;
Y is --CH.sub.2 --, CO, SO.sub.2 --, or a bond;
R.sub.1 and R.sub.2 are independently selected from the following: branched
or unbranched C.sub.1-6 alkyl-, C.sub.1-6 alkyloxy-, --N--C.sub.1-6
alkyl-, C.sub.3-8 cycloalkyl-, phenyl, naphthyl, pyridyl, furanyl,
thienyl, or quinolinyl, any of which may be substituted once or twice with
C.sub.1-5 alkyl, C.sub.3-8 cycloalkyl, phenyl, quinolinyl, pyridyl,
furanyl, thienyl, C.sub.1-6 -alkoxy, Br, F, or Cl.
The compounds of the present invention may have asymmetric centers and
occur as racemates, racemic mixtures and as individual diastereomers,
enantiomers, or mixtures of enantiomers with all isomeric forms being
included in the present invention.
When any variable (e.g., R.sub.1 or R.sub.2, etc.) occurs more than one
time in any constituent or in Formula I, its definition on each occurrence
is independent of its definition at every other occurrence. Also,
combinations of substituents and/or variables are permissible only if such
combinations result in stable compounds.
As used herein except where noted, "alkyl" is intended to include both
branched- and straight-chain saturated aliphatic hydrocarbon groups having
the specified number of carbon atoms (Me is methyl, Et is ethyl, Pr is
propyl, Bu is butyl); "alkoxy" represents an alkyl group of indicated
number of carbon atoms attached through an oxygen bridge. "Halo", as used
herein, means fluoro, chloro, bromo or iodo.
The pharmaceutically-acceptable salts of the compounds of Formula I (in the
form of water- or oil-soluble or dispersible products) include the
conventional non-toxic salts or the quaternary ammonium salts of these
compounds, which are formed, e.g., from inorganic or organic acids.
Examples of such acid addition salts include acetate, adipate, alginate,
aspartate, benzoate, bisulfate, citrate, digluconate, dodecylsulfate,
fumarate, glycerophosphate, hemisulfate, hydrochloride,
2-hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate, succinate
and tartrate.
One preferred embodiment of this invention is compounds of Formula I,
wherein
Y is --CH.sub.2 --;
R.sub.1 is
(i) phenyl, unsubstituted or substituted with halo; or
(ii) cyclohexyl;
R.sub.2 is
(i) H or
(ii) benzyl, unsubstituted or substituted with halo; or pharmaceutically
acceptable salt, hydrate or ester thereof.
Preferred compounds of this invention include the following:
Compound A:
##STR2##
named 4-[N-benzyl-3-(4-chlorobenzyl)piperidin-3-yl]2,4-dioxobutanoic acid
hydrochloride,
or pharmaceutically acceptable salt, hydrate or ester thereof;
Compound B:
##STR3##
named 4-[N-benzyl-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic acid
hydrochloride,
or pharmaceutically acceptable salt, hydrate or ester thereof;
Compound C:
##STR4##
named
4-N-(p-chlorobenzyl)-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic
acid hydrochloride,
or pharmaceutically acceptable salt, hydrate or ester thereof;
Compound D:
##STR5##
named
4-[1-cyclohexylmethyl-4-(p-chlorobenzyl)piperidin-4-yl]2,4-dioxobutanoic
acid hydrochloride,
or pharmaceutically acceptable salt, hydrate or ester thereof.
The compounds of the present invention are prepared in accordance with
Schemes I-III.
##STR6##
According to Scheme I, the N-protected ester 1 is alkylated with the
appropriate halide, e.g., 4-chlorobenzyl chloride, in the presence of an
amide base such as LDA or LiHMDS [lithium bis(trimethylsilyl)-amide]. The
alkylated ester product 2 is hydrolyzed to a carboxylic acid 3. Reaction
with carbonyldiimidazole affords the corresponding substituted acyl
imidazole, which reacts with the appropriate amine, e.g.,
N,O-dimethylhydroxylamine, to give amide 4. Alkylation with Grignard
reagent followed by deprotection of the nitrogen results in 6. Amine 6 is
alkylated by reaction with a slight excess of halide in base to afford 7.
Reaction with an excess of reducing agent and the appropriate oxalic acid
gives 8.
Scheme I is illustrated by Example 1, but is not limited to this particular
example.
##STR7##
For 3-substituted piperidine compounds of Formula I, Scheme II illustrates
a method of synthesis. In Step C, the preferred reagent is oxalylchloride
instead of carbonyldiimidazole. Otherwise, synthesis of 3-substituted
piperidine compounds of Formula I substantially resembles the synthesis of
4-substituted piperidine compounds of Formula I as set forth in Scheme I.
Scheme 2 is specifically illustrated by Example 2, but is not limited to
this particular example.
##STR8##
In Scheme Ill, 4-sulfonyl piperdine compounds of Formula I are readily
prepared by variations of Scheme I. The sulfonyl substituent can be added
first to give, for example, 17. This variation eliminates the steps of
deprotection and alkylation of the piperidinyl nitrogen (Steps E and F of
scheme I).
Scheme 3 is specifically illustrated by Example 3, but is not limited to
this particular example.
The compounds invention include but are not limited to those of the
following Tables 1, 2 and 3:
TABLE 1
______________________________________
##STR9##
##STR10##
Example R MP (.degree.C.)
______________________________________
2
##STR11## 87-90
3
##STR12## 168-170
4
##STR13## 178-180
5
##STR14## 60-62
6
##STR15## 171-173
7
##STR16## 185-186
8
##STR17## 215-218
9
##STR18## 125-131
10
##STR19## 160-162
11
##STR20## 135-140
12
##STR21## 176-178
13
##STR22## 195-197
14
##STR23## 206-208
15
##STR24## 133-136
16
##STR25## 198-200
17
##STR26## 178-180
18
##STR27## >275
19
##STR28## 120-122
______________________________________
TABLE 2
______________________________________
##STR29##
##STR30##
Example R MP (.degree.C.)
______________________________________
21
##STR31## 160-162
22
##STR32## 55-57
23
##STR33## 70-73
24
##STR34## 168-170
25
##STR35## 148-152
26
##STR36## 190-192
27
##STR37## 189-191
28
##STR38## 184-187
29
##STR39## 117-119
##STR40##
##STR41##
30
##STR42## 74-77
31
##STR43## 154-155
32
##STR44## 120-125
33
##STR45## 70-73
34
##STR46## 209-211
35
##STR47## 130--133
36
##STR48## 108-110
37
##STR49## 167-169
38
##STR50## 212-215
______________________________________
TABLE 3
______________________________________
##STR51##
Example R MP (.degree.C.)
______________________________________
40
##STR52## 85-90
41
##STR53## 154-157
42
##STR54## 62-66
43
##STR55## 75-80
44
##STR56## 130-134
45
##STR57## 95-100
46
##STR58## 65-70
47
##STR59## 164-167
48
##STR60## 114-120
49
##STR61## 100-105
##STR62##
50
##STR63## 114-120
51
##STR64## 178-180
52
##STR65## 85-95
52
##STR66## 120-130
54
##STR67## 172-174
55
##STR68## 98-100
56
##STR69## 55-60
57
##STR70## 89-91
58
##STR71## 95-97
______________________________________
The compounds of the present invention are useful in the inhibition of
influenza virus cap-dependent endonuclease, and the prevention or
treatment of infection by the influenza virus.
The compounds of this invention are also useful in the preparation and
execution of screening assays for antiviral compounds. For example, the
compounds of this invention are useful for isolating enzyme mutants, which
are excellent screening tools for more powerful antiviral compounds.
Furthermore, the compounds of this invention are useful in establishing or
determining the binding site of other antivirals to influenza virus
cap-dependent endonuclease, e.g., by competitive inhibition. Thus the
compounds of this invention are commercial products to be sold for these
purposes.
For these purposes, the compounds of the present invention may be
administered orally, parenterally (including subcutaneous injections,
intravenous, intramuscular, intrasternal injection or infusion
techniques), by inhalation spray, or rectally, in dosage unit formulations
containing conventional non-toxic pharmaceutically-acceptable carriers,
adjuvants and vehicles.
Thus, in accordance with the present invention there is further provided a
method of treating and a pharmaceutical composition for treating influenza
virus infection and influenza. The treatment involves administering to a
patient in need of such treatment a pharmaceutical composition comprising
a pharmaceutical carder and a therapeutically-effective mount of a
compound of the present invention, or a pharmaceutically-acceptable salt
thereof.
These pharmaceutical compositions may be in the form of
orally-administrable suspensions or tablets; nasal sprays; sterile
injectable preparations, for example, as sterile injectable aqueous or
oleagenous suspensions or suppositories.
When administered orally as a suspension, these compositions are prepared
according to techniques well-known in the art of pharmaceutical
formulation and may contain microcrystalline cellulose for imparting bulk,
alginic acid or sodium alginate as a suspending agent, methylcellulose as
a viscosity enhancer, and sweeteners/flavoring agents known in the art. As
immediate release tablets, these compositions may contain microcrystalline
cellulose, dicalcium phosphate, starch, magnesium stearate and lactose
and/or other excipients, binders, extenders, disintegrants, diluents and
lubricants known in the art.
When administered by nasal aerosol or inhalation, these compositions are
prepared according to techniques well-known in the art of pharmaceutical
formulation and may be prepared as solutions in saline, employing benzyl
alcohol or other suitable preservatives, absorption promoters to enhance
bioavailability, fluorocarbons, and/or other solubilizing or dispersing
agents known in the art.
The injectable solutions or suspensions may be formulated according to
known an, using suitable non-toxic, parenterally-acceptable diluents or
solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or
isotonic sodium chloride solution, or suitable dispersing or wetting and
suspending agents, such as sterile, bland, fixed oils, including synthetic
mono- or diglycerides, and fatty acids, including oleic acid.
When rectally administered in the form of suppositories, these compositions
may be prepared by mixing the drug with a suitable non-irritating
excipient, such as cocoa butter, synthetic glyceride esters or
polyethylene glycols, which are solid at ordinary temperatures, but
liquidify and/or dissolve in the rectal cavity to release the drug.
Dosage levels of the order of 0.02 to 5.0 or 10.0 grams-per-day are useful
in the treatment or prevention of the above-indicated conditions, with
oral doses two-to-five times higher. For example, infection by influenza
virus is effectively treated by the administration of from 10 to 50
milligrams of the compound per kilogram of body weight from one to three
times per day. It will be understood, however, that the specific dose
level and frequency of dosage for any particular patient may be varied and
will depend upon a variety of factors including the activity of the
specific compound employed, the metabolic stability and length of action
of that compound, the age of the patient, body weight, general health,
sex, diet, mode and time of administration, rate of excretion, drug
combination, the severity of the particular condition, and the host
undergoing therapy.
The present invention is also directed to combinations of the influenza
virus inhibitory compounds with one or more agents useful in the treatment
of influenza. For example, the compounds of this invention may be
effectively administered, whether at periods of pre-exposure and/or
post-exposure, in combination with effective amounts of the influenza
virus antivirals, immunomodulators, anti-infectives, or vaccines known to
those of ordinary skill in the art.
EXAMPLE 1
4-[N-Benzy-4-(4-Chlorobenzyl)-piperidin-4-yl]-2,4-dioxobutanoic acid
Hydrochloride
##STR72##
Step A: Ethyl-4-(4-Chlorobenzyl)-N-Boc-isonipecotate
A solution of ethyl-N-boc-isonipecotate (51.4 g, 0.2 mole) in
tetrahydrofuran (1 L) at -78.degree. C. was treated with a solution of
lithium bis (trimethylsilyl)amide in tetrahydrofuran (220 mL of a 1M
solution). The solution was stirred at -60.degree. for 15 minutes at which
time 4-chlorobenzyl chloride (33.8 g, 0.22 moles) was added and the
reaction warmed to room temperature over 1.5 hours. The reaction was
concentrated at reduced pressure to one quarter volume and then poured
into saturated aqueous sodium bicarbonate (1 L) and extracted with ethyl
acetate (2.times.800 mL). The combined organic extracts were dried over
anhydrous magnesium sulfate, filtered, and concentrated at reduced
pressure. The residue was chromatographed on silica gel with 30% ethyl
acetate/hexane as eluent to give 61.3 g. .sup.1 H NMR CDCl.sub.3 .delta.
7.22 (d, J=8.0 Hz, 2H), 6.97 (d, J=8.0 Hz, 2H), 4.10 (q, J=7 Hz, 2H), 3.90
(m, 2H), 2.8 (m, 2H), 2.8 (s, 2H), 2.05 (m, 2H), 1.44 (s, 9H), 1.40-1.30
(m, 2H), 1.15 (t, J=t Hz, 1H).
Step B: 4-(4-Chlorobenzyl)-N-boc-isonipecotic acid
The material thus obtained in Step A was dissolved in isopropyl alcohol
(150 mL) and tetrahydrofuran (150 mL) and treated with 10N NaOH (250 mL).
The mixture was heated at reflux for 48 hr. The reaction was cooled to
room temperature and carefully neutralized by pouring over 1 L of crushed
ice and adding 6N HCl until pH 3. The mixture was then extracted with
ethyl acetate (3.times.500 mL). The combined organic extracts were dried
over anhydrous magnesium sulfate, filtered, and concentrated at reduced
pressure to give 16 g of the product. .sup.1 H NMR CDCl.sub.3 .delta. 7.25
(d, J=8.5 Hz, 2H), 7.04 (d, J=8.5 Hz, 2H), 3.95 (m, 2H), 2.9 (m, 2H), 2.84
(s, 2H), 2.05 (m, 2H), 1.45 (s, 9H), 1.45-1.30 (m, 2H).
Step C: N-boc4-(4-Chlorobenzyl)-piperidine-4-carboxylic
acid-(N,O-Dimethylmethylhydroxamide)
A solution of 4-(4-chlorobenzyl-N-boc-piperidine-4-carboxylic acid (52 g,
0.131 moles) in N,N-dimethylformamide (100 mL) at room temperature was
treated with carbonyldiimidazole (25.7 g, 0.158 mol) and warmed to
60.degree. C. for 15 minutes. N,O-dimethyl-hydroxylamine hydrochloride
(30.8 g, 0.316 mole) was then added and the reaction stirred at 60.degree.
C. for 15 minutes. The reaction was then cooled to room temperature and
aged for 1 hour. After stirring for 1 hour at room temperature the
reaction was poured into saturated aqueous sodium bicarbonate and
extracted with ethyl acetate (3.times.300 mL). The combined organic
extracts were dried over anhydrous magnesium sulfate, filtered, and
concentrated at reduced pressure to give 58 g of the title compound.
.sup.1 H NMR CDCl.sub.3 .delta. 7.20 (d, J=8.5 Hz, 2H), 6.98 (d, J=8.5 Hz,
2H), 3.93 (m, 2H), 3.70 (s, 3H), 3.19 (s, 3H), 2.98 (m, 2H), 2.96 (s, 2H),
2.21 (m, 2H), 1.45 (s, 9H), 1.45-1.30 (m, 2H).
Step D: N-boc-4-acetyl-4-(4-Chlorobenzyl)-piperidine
A solution of N-boc-4-(4-Chlorobenzyl)-piperidine-4-carboxylic
acid-(N,O-Dimethylmethylhydroxamide) (48 g, 0.120 mol) in tetrahydrofuran
(400 mL) was treated with a solution of methylmagnesium bromide (60 mL of
a 3M solution in tetrahydrofuran, 0.18 mol) and heated to reflux for
thirty minutes. The reaction was then cooled to room temperature, poured
into saturated aqueous sodium bicarbonate (1.5 L) and extracted with ethyl
acetate (3.times.500 mL). The combined organic extracts were dried over
anhydrous magnesium sulfate, filtered, and concentrated at reduced
pressure to give 25.2 g of the title compound. .sup.1 H NMR CDCl.sub.3
.delta. 7.22 (d, J=8.5 Hz, 2H), 6.96 (d, J=8.5 Hz, 2H), 3.93 (m, 2H), 2.92
(m, 2H), 2.89 (s, 2H), 2.10 (s, 3H), 2.05 (m, 2H), 1.45 (s, 9H), 1.50-1.30
(m, 2H).
Step E: 4-Acetyl-4-(4-Chlorobenzyl)-piperidine hydrochloride
HCl gas was bubbled into a solution of
N-boc-4-acetyl-4-(4-chlorobenzyl)-piperidine (25 g, 0.071 mol) in ethyl
acetate (300 mL) for 15 minutes. The reaction was concentrated at reduced
pressure and the solid collected to give 20.3 g of product. .sup.1 H NMR
d6 DMSO .delta. 9.1 (br s, 1H), 8.8 (br s, 1H), 7.30 (d, J=8.0 Hz, 2H),
6.96 (d, J=8.0 Hz, 2H), 3.13 (m, 2H), 2.92 (s, 2H), 2.65 (m, 2H), 2.13 (s,
3H), 2.05 (m, 2H), 1.70 (m, 2H).
Step F: N-Benzyl-4-Acetyl4-(4-Chlorobenzyl)-piperidine
A suspension of 4-Acetyl-4-(4-chlorobenzyl)-piperidine hydrochloride (0.600
g, 2.09 mmol) in acetonitrile (20 mL) was treated with solid sodium
bicarbonate (200 mg, xs) and benzyl chloride (292 mg, 2.3 mol) and heated
to reflux for 3 hours. The reaction was then cooled to room temperature,
poured into saturated aqueous sodium bicarbonate (200 mL) and extracted
with ethyl acetate (3.times.100 mL). The combined extracts were dried over
anhydrous magnesium sulfate, filtered, and concentrated at reduced
pressure. The residue was chromatographed on silica gel eluting with 2%
Methanol/chloroform to give 706 mg of the title compound. .sup.1 H NMR
CDCl.sub.3 .delta. 7.25 (m, 5H), 7.22 (d, J=8.5 Hz, 2H), 6.96 (d, J=8.5
Hz, 2H), 3.45 (s, 2H), 2.78 (s 2H), 2.76 (m, 2H), 2.20-2.0 (m, 4H), 2.06
(s, 3H), 1.75-1.50 (m, 2H).
Step G: 4-[N-Benzyl-4-(4-Chlorobenzyl)-piperidin-4-yl]-2,4-dioxo-butanoic
acid Hydrochloride
A solution of N-Benzyl-4-Acetyl-4-(4-Chlorobenzyl)-piperidine (600 mg, 1.75
mmol) and dimethyl oxalate (310 mg, 2.63 mmol) in dimethoxyethane (20 mL)
was treated with sodium hydride (105 mg of a 60% dispersion in mineral
oil, 2.63 mmol) and heated to reflux for 3 hours. The reaction was then
cooled to room temperature, poured into 1 N aqueous HCl (200 mL) and
extracted with ethyl ether (1.times.50 mL). The ether extract was
discarded. The pH of the aqueous phase was adjusted to pH 8 and extracted
with ethyl acetate (3.times.200 mL). The combined extracts were dried over
anhydrous magnesium sulfate, filtered, and concentrated at reduced
pressure. The crude ester thus obtained was dissolved in THF (5 mL) and 3
N HCl (20 mL) and heated to reflux for 1 hour. The reaction was then
cooled to room temperature, and concentrated at reduced pressure. The
residue was triturated with tetrahydrofuran and the solid collected by
filtration and crystallized from isopropanol to give 200 mg of the title
compound. mp. 189.degree.-181.degree. C.
Elemental analysis for C.sub.23 H.sub.24 ClNO.sub.4.HCI.IPA
Calculated: C, 60.64; H, 6.56; N, 2.72
Found: C, 60.61; H, 6.46; N, 2.65
EXAMPLE 2
4-[N-Benzyl-3-(4-Chlorobenzyl)-piperidin-3-yl]-2,4-dioxobutanoic acid
Hydrochloride
##STR73##
Step A: Ethyl-3-(4-chlorobenzyl)-N-Boc-piperidin-3-carboxylate
A solution of ethyl-N-boc-nipecotate (45 g, 0.18 mole) in tetrahydrofuran
(200 mL) at -40.degree. C. was treated with a solution of lithium bis
(trimethylsilyl)amide in tetrahydrofuran (320 mL of a 1M solution). The
solution was stirred at -40.degree. for 15 minutes at which time
4-Chlorobenzyl chloride (34.59 g, 0.215 moles) was added and the reaction
warmed to room temperature over 1.5 hours. The reaction was poured into
saturated aqueous sodium bicarbonate (1 L) and extracted with ethyl
acetate (3.times.500 mL). The combined organic extracts were dried over
anhydrous magnesium sulfate, filtered, and concentrated at reduced
pressure. The residue was chromatographed on silica gel with 30% ethyl
acetate/hexane as eluent to give 36.5 g. .sup.1 H NMR CDCl.sub.3 .delta.
7.22 (d, J=8.5 Hz, 2H), 7.05 (d, J=8.5 Hz, 2H), 4.1-3.8 (m, 3H), 3.55 (m,
1H), 3.15 (m, 1H), 2.9 (d, J=13 Hz, 1H), 2.7 (d, J=13 Hz, 1H), 2.00 (m,
1H), 1.80-1.40 (m, 3H), 1.44 (s, 9H), 1.15 (t, J=7 Hz, 3H).
Step B: N-boc-3-(4-chlorobenzyl)piperidin-3-carboxylic acid
A suspension of Ethyl-3-(4-chlorobenzyl)-N-Boc-piperidin-3-caroxylate (36.4
g, 0.105 mol) in isopropyl alcohol (300 mL) was treated with 10N NaOH (300
mL). The mixture was heated at reflux for 48 hours. The reaction was
cooled to room temperature and carefully neutralized by pouting over 1 L
of crashed ice and adding 6 N HCl until pH 3. The mixture was then
extracted with ethyl acetate (3.times.500 mL). The combined organic
extracts were dried over anhydrous magnesium sulfate, filtered, and
concentrated at reduced pressure to give 31 g of the product. .sup.1 H NMR
CDCl.sub.3 .delta. 10.18 (br s), 7.26 (d, J=8.5 Hz, 2H), 7.04 (d, J=8.5
Hz, 2H), 3.95 (m, 1H), 3.6 (m, 1H), 3.2 (m, 2H), 2.90 (d, J=12 Hz, 1H),
2.78 (d, J=12 Hz, 1H), 2.00 (m, 1H), 1.75-1.30 (m, 3H), 1.44 (s, 9H).
Step.C: N-boc-3-(4-Chlorobenzyl)-piperidine-3-carboxylic
acid-(N,O-Dimethylmethylhydroxamide)
A solution of 3-(4-chlorobenzyl)-N-boc-nipecotic acid (23 g, 0.064 moles)
in methylene chloride (500 mL) at room temperature was treated with DMF (2
mL) and oxalyl chloride (8.5 mL), and stirred at room temperature for 1
hr. The reaction was then concentrated at reduced pressure and the crude
acid chloride redissolved in methylene chloride (500 mL). A suspension of
N,O-dimethylhydroxylamine hydrochloride (12.68 g, 0.129 mole) in pyridine
(50 mL) was then added and the reaction stirred at room temperature for
one hour. The reaction was then poured into saturated aqueous sodium
bicarbonate (1) and extracted with ethyl acetate (3.times.300 mL). The
combined extracts were dried over anhydrous magnesium sulfate, filtered,
and concentrated at reduced pressure to give 26.1 g of the title compound.
.sup.1 H NMR CDCl.sub.3 .delta. 7.15 (d, J=8.5 Hz, 2H), 7.04 (d, J=8.5 Hz,
2H), 3.95-3.6 (m, 2H), 3.75 (br s, 3H), 3.2-3.0 (m, 2H), 3.0 (s, 3H),
2.98-2.6 (m, 2H), 2.00-1.5 (m, 4H), 1.40 (s, 9H).
Step D: N-Boc-3-acetyl-3-(4-Chlorobenzyl)-piperidine
A solution of N-boc-3-(4-Chlorobenzyl)-piperidine-3-carboxylic
acid-(N,O-Dimethylmethylhydroxamide) (25.59 g, 0.064 mol) in
tetrahydrofuran (200 mL) was treated with a solution of methylmagnesium
bromide (64.5 mL of a 3M solution in tetrahydrofuran, 0.194 mol) and
heated to reflux for thirty minutes. The reaction was then cooled to room
temperature poured into saturated aqueous sodium bicarbonate (1 L) and
extracted with ethyl acetate (3.times.500 mL). The combined extracts were
dried over anhydrous magnesium sulfate, filtered, and concentrated at
reduced pressure to give 12.2 g of the title compound. .sup.1 H NMR
CDCl.sub.3 .delta. 7.25 (d,=8.5 Hz, 2H), 7.00 (d,=8.5 Hz, 2H), 4.3 (m,
1H), 3.7 (m, 2H), 3.3-2.6 (m, 5H), 2.10-2.00 (m, 1H), 2.05 (s, 3H),
1.70-1.20 (m, 3H), 1.45 (s, 9H).
Step E: 3-Acetyl-3-(4-Chlorobenzyl),piperidine hydrochloride
HCl gas was bubbled into a solution of
N-boc-3-acetyl-3-(4-Chlorobenzyl)-piperidine (20.46 g, 0.058 mol) in ethyl
acetate (300 mL) for 15 minutes. The reaction was concentrated at reduced
pressure and the solid collected to give 16 g of product. .sup.1 H NMR d6
DMSO .delta. 9.1 (br s, 1H), 8.8 (br s, 1H), 7.30 (d,=8.0 Hz, 2H), 6.96
(d,=8.0 Hz, 2H), 3.13 (m, 2H), 2.92 (s, 2H), 2.65 (m, 2H), 2.13 (s, 3H),
2.05 (m, 2H), 1.70 (m, 2H).
Step F: N-Benzyl-3-acetyl-3-(4-chlorobenzyl)-piperidine
A suspension of 3-Acetyl-3-(4-Chlorobenzyl)-piperidine hydrochloride (0.7
g, 2.43 mmol) in acetonitrile (20 mL) was treated with solid sodium
bicarbonate (200 mg, 4.8 mmol) and benzyl chloride (369 mg, 2.9 mol) and
heated to reflux for 2 hours. The reaction was then cooled to room
temperature, poured into saturated aqueous sodium bicarbonate (200 mL) and
extracted with ethyl acetate (3.times.100 mL). The combined organic
extracts were dried over anhydrous magnesium sulfate, filtered, and
concentrated at reduced pressure. The residue was chromatographed on
silica gel eluting with 2% methanol/chloroform to give 492 mg of the title
compound. .sup.1 H NMR CDCl.sub.3 .delta. 7.40-7.25 (m, 5H), 7.22 (d,=8.5
Hz, 2H), 6.90 (d,=8.5 Hz, 2H), 3.45 (m, 2H), 3.00 (m, 1H), 2.80-2.50 (m,
3H), 2.20-1.90 (m, 4H), 2.00 (s, 3H), 1.60-1.50 (m, 2H), 1.30-1.10 (m,
1H).
Step G: 4-[N-Benzyl-3-(4-chlorobenzyl)-piperidin-3-yl]-2,4-dioxobutanoic
acid Hydrochloride
A solution of N-Benzyl-3-acetyl-3-(4-chlorobenzyl)-piperidine (492 mg, 1.44
mmol) and dimethyl oxalate (201 mg, 1.7 mmol) in dimethoxyethane (20 mL)
was treated with sodium hydride (140 mg of a 60% dispersion in mineral
oil, 3.4 mmol) and heated to reflux for 5 hours. The reaction was then
cooled to room temperature, poured into saturated sodium bicarbonate (100
mL) and extracted with ethyl acetate (3.times.100 mL). The combined
extracts were dried over anhydrous magnesium sulfate, filtered, and
concentrated at reduced pressure. The crude ester thus obtained was
purified by chromato-graphy on silica gel eluting with 10%
methanol/chloroform. The material thus obtained was dissolved in
tetrahydrofuran (5 mL) and 3N HCl (20 mL) and heated to reflux for 1 hour.
The reaction was then cooled to room temperature, and concentrated at
reduced pressure. The residue was triturated with tetrahydrofuran and the
solid collected by filtration and crystallized from isopropanol to give 35
mg of the title compound. mp. 160.degree.-165.degree. C.
Elemental analysis for C.sub.23 H.sub.24 ClNO.sub.4.HCI
Calculated: C, 61.34; H, 5.60; N, 3.11
Found: C, 61.34; H, 5.87; N, 3.39
EXAMPLE 3
4-[N-Benzenesulfonyl-4-(benzyl)-piperidin-4-yl]-2,4-dioxobutanoic acid
##STR74##
Step A: Ethy-N-benzenesulfonyl-piperidine-4-carboxylate
A solution of ethyl isonipecotate (15.7 g, 0.1 mol) in methylene chloride
(100 mL) at 0.degree. C. was treated with pyridine (10 mL) and then
benzenesulfonyl chloride (17.6 g, 0.1 mol). The reaction was warmed to
room temperature and stirred at room temperature for 2 hours. The reaction
was then poured into 1N HCl (1 L) and extracted with ethyl acetate
(2.times.500 mL). The combined extracts were dried over anhydrous
magnesium sulfate, filtered, and concentrated at reduced pressure. The
solid was recrystallized from ethyl acetate to give 17 g of product.
.sup.1 H NMR CDCl.sub.3 .delta. 7.78 (d, J=6.0 Hz, 2H), 7.65-7.50 (m, 3H),
4.1 (q, J=6.8 Hz, 2H), 3.62 (m, 2H), 2.50 (dt, J=3, 11.5 Hz, 2H), 2.25 (m,
1H), 2.03-1.90 (m, 2H), 1.85-1.75 (m, 2H), 1.33 (t, J=6.8 Hz, 3H).
Step B: Ethyl-N-Benzenesulfonyl-4-(benzyl)-piperidine-4-carboxylate
A solution of ethyl-N-benzenesulfonyl-isonipecotate (5 g, 0.016 mole) in
tetrahydrofuran (1 L) at -78.degree. C. was treated with a solution of
Lithium bis (trimethylsilyl)amide in tetrahydrofuran (18.5 mL of a 1M
solution). The solution was stirred at -60.degree. for 15 minutes at which
time benzyl bromide (3.15 g, 0.016 moles) was added and the reaction
warmed to room temperature over 1.5 hours. The reaction was concentrated
at reduced pressure to @ one quarter volume and then poured into saturated
aqueous sodium bicarbonate (1 L) and extracted with ethyl acetate
(2.times.800 mL). The combined extracts were dried over anhydrous
magnesium sulfate, filtered, and concentrated at reduced pressure. The
residue was chromatographed on silica gel with 30% ethyl acetate/hexane as
eluent to give 5.1 g. .sup.1 H NMR CDCl.sub.3 .delta. 7.7 (d,=8.5 Hz, 2H),
7.6-7.4 (m, 3H), 7.3-7.2 (m, 3H), 7.1-7.0 (m, 2H), 3.98 (q, J=7 Hz, 2H),
3.65 (m, 2H), 2.78 (s, 2H), 2.35 (dt, J=2.5, 12 Hz, 2H), 2.19 (d, J=12 Hz,
2H), 1.61 (dt, J=4.5, 12 Hz, 2H).
Step C: N-Benzenesulfonyl-4-(benzyl)-piperidine-4-carboxylic acid
The material thus obtained was dissolved in isopropyl alcohol
tetrahydrofuran (150 mL) and treated with 6N NaOH (250 mL). The mixture
was heated to reflux for 48 hours. The reaction was cooled to room
temperature and carefully neutralized by pouring over 1 L of crashed ice
and adding 6N HCl until pH 3. The mixture was then extracted with ethyl
acetate (3.times.500 mL). The combined extracts were dried over anhydrous
magnesium sulfate, filtered, and concentrated at reduced pressure to give
4.1 g of the product. .sup.1 H NMR d6 DMSO .delta. 7.70-7.60 (m, 5H),
7.30-7.15 (m, 3H), 7.06 (m, 2H), 3.55 (m, 2H), 2.72 (s, 2H), 2.15 (app t,
J=12 Hz, 2H), 1.98 (app d, J=12 Hz, 2H), 1.50 (app dt, J=2,12 Hz, 2H).
Step D: N-Benzenesulfonyl-4-(benzyl)-piperidine-4-carboxylic
acid-(N,O-Dimethylmethylhydroxamide)
A solution of N-benzenesulfonyl-4-(4-chlorobenzyl)-piperidine-4-carboxylic
acid (52 g, 0.131 moles) in DMF (100 mL) at room temperature was treated
with carbonyldiimidazole (25.7 g, 0.158 mol mL) and warmed to 60.degree.
C. for 15 minutes. N,O-dimethylhydroxylamine hydrochloride (30.8 g, 0.316
mole) was then added and the reaction stirred at 60.degree. C. for 15
minutes. The reaction was then cooled to room temperature and aged for 1
hour. After stirring for 1 hour at room temperature the reaction was
poured into saturated aqueous sodium bicarbonate and extracted with ethyl
acetate (3.times.300 mL). The combined extracts were dried over anhydrous
magnesium sulfate, filtered, and concentrated at reduced pressure to give
58 g of the title compound. .sup.1 H NMR CDCl.sub.3 .delta. 7.20 (d,=8.5
Hz, 2H), 6.98 (d,=8.5 Hz, 2H), 3.93 (m, 2H), 3.70 (s, 3H), 3.19 (s, 3H),
2.98 (m, 2H), 2.96 (s, 2H), 2.21 (m, 2H), 1.45 (s, 9H), 1.45-1.30 (m, 2H).
Step E: N-Benzenesulfonyl-4-acetyl-4-(benzyl)-piperidine
A solution of N-Benzenesulfonyl-4-(4-chlorobenzyl)-piperidine-4-carboxylic
acid-(N,O-Dimethylmethylhydroxamide) (48 g, 0.120 mol) in tetrahydrofuran
(400 mL) was treated with a solution of methylmagnesium bromide (60 mL of
a 3N solution in tetra-hydrofuran, 0.18 mol) and heated to reflux for
thirty minutes. The reaction was then cooled to room temperature, poured
into saturated aqueous sodium bicarbonate (1.5 L) and extracted with ethyl
acetate (3.times.500 mL). The combined extracts were dried over anhydrous
magnesium sulfate, filtered, and concentrated at reduced pressure to give
25.2 g of the title compound. .sup.1 H NMR CDCl.sub.3 .delta. 7.22 (d,=8.5
Hz, 2H), 6.96 (d,=8.5 Hz, 2H), 3.93 (m, 2H), 2.92 (m, 2H), 2.89 (s, 2H),
2.10 (s, 3H), 2.05 (m, 2H), 1.45 (s, 9H), 1.50-1.30 (m, 2H).
Step F: 4-[N-Benzenesulfonyl-4-(benzyl)-piperidin-4-yl]-2,4-dioxobutanoic
acid
A solution of N-Benzyl-4-acetyl-4-(4-chlorobenzyl)-piperidine (600 mg, 1.75
mmol) and dimethyl oxalate (310 mg, 2.63 mmol) in dimethoxyethane (20 mL)
was treated with sodium hydride (105 mg of a 60% dispersion in mineral
oil, 2.63 mmol) and heated to reflux for 3 hours. The reaction was then
cooled to room temperature, poured into 1N aqueous HCl (200 mL) and
extracted with ethyl ether (1.times.50 mL). The ether extract was
discarded. The pH of the aqueous phase was adjusted to pH 8 and extracted
with ethyl acetate (3.times.200 mL). The combined extracts were dried over
anhydrous magnesium sulfate, filtered, and concentrated at reduced
pressure. The crude ester thus obtained was dissolved in tetrahydrofuran
(5 mL) and 3N NaOH (20 mL) and stirred at room temperature for 1 hour. The
reaction was then cooled to room temperature, and concentrated at reduced
pressure. The residue was triturated with THF and the solid collected by
filtration and crystallized from isopropanol to give 200 mg of the title
compound. mp. 189.degree.-181.degree. C.
Elemental analysis for C.sub.23 H.sub.24 ClNO.sub.4.HCl.IPA
Calculated: C, 60.64; H, 6.56; N, 2.72
Found: C, 60.61; H, 6.46; N, 2.65
EXAMPLE 4
SUMMARY BIOLOGICAL DATA FOR DIOXOBUTANOIC ACIDS
I. IDENTIFICATION OF IN VITRO INHIBITORY ACTIVITY
The dioxobutanoic acid inhibitors were identified in an in vitro screen
which was established for the identification of inhibitors of influenza
primary transcription (FLUTIN) in our laboratory. The assay consisted of
detergent-disrupted influenza virions as the enzyme source to which was
added a capped and methylated primer, alfalfa mosaic virus (ALMV) RNA
segment 4, and ribonucleotide triphosphates, one of which was radiolabeled
to measure the incorporation into RNA by TCA precipitation. The inhibitory
compounds had IC.sub.50 s ranging from 0.2-29.0 .mu.M in influenza
transcription.
II. SPECIFIC PROTOCOLS
a. ALMV-primed flu transcription-Samples or DMSO were incubated for 60 min
at 31.degree. C. in a final reaction mixture containing 20 ng/.mu.l flu
APR8 virus; 2 ng/.mu.l ALMV capped primer (880 nt); 2 ng/.mu.l tRNA; 100
.mu.M ATP, 50 .mu.M C and GTP, 1 .mu.M UTP and 0.3 .mu.M [35]S-UTP in 100
mM Tris-HCl, pH 7.8/0.25% Triton-N-100/100 mM KCl/5 mM MgCl.sub.2 /1 mM
DTT. Reaction product was quantitated by TCA precipitation on glass fiber
filters, followed by liquid scintillation counting.
b. ApG-primed flu transcription-Samples or DMSO were incubated in a fmal
reaction mixture as in a. above, with 200 .mu.M ApG replacing ALMV primer.
c. VSV transcription-Samples or DMSO were incubated for 60 min at
31.degree. C. in a final reaction mixture containing 20 ng/.mu.l of VSV; 2
ng/.mu.l of tRNA; 100 .mu.M ATP; 50 .mu.M C and GTP; 1 .mu.M UTP and 0.3
.mu.M [35]S-UTP; in 50 mM Tris-HCl, pH 8.0/100 mM NaCl/4 mM DTT/0.05%
Triton-N-100/5 mM MgCl.sub.2. Reaction product was quantitated by TCA
precipitation onto glass fiber filters, followed by liquid scintillation
counting.
d. Hela RNA polymerase II-Compounds or DMSO were incubated in a run-off
transcription assay using Hela RNA polymerase II in Hela extract with 50
ng/.mu.l of pD5 template (Adeno major late promoter) in a final reaction
containing 500 .mu.M A,C,GTP and 0.5 .mu.M [32]P-UTP in 15 mM Tris-HCl, pH
7.9/7.0 mM MGCl.sub.2 /32 mM (NH.sub.4).sub.2 SO.sub.4 /0.2 mM EDTA/1.3 mM
DTT for 60 min at 30.degree. C. Following ethanol precipitation, reaction
products were electrophoresed on 8% polyacrylamide gels containing 7M
urea.
e. 13, 22, 70 nt-primed flu transcription assays-Samples or DMSO were
tested in transcription as in (a) above with purified polymerase cores at
a concentration of 2 ng/.mu.l replacing APR8 virus and primed with 10 nM
synthetic ALMV capped primers (13, 22, 70 nt) replacing ALMV primer (880
nt) for 40 min. Reaction was quantitated by TCA precipitation.
f. Flu Cleavage-Samples or DMSO were tested in a final reaction mixture
containing 2.5 ng/.mu.l of purified polymerase cores; 1 ng/.mu.l of tRNA;
0.5 .mu./.mu.l of RNasin; and 5.5 nM of [32]P-radiolabeled ALMV in 50 mM
Tris-HCl, pH 7.8/100 mM KCl/5 mM MgCl.sub.2 /1 mM DTT for 30 min. The
reaction was stopped by addition of an equal volume of 95% formamide
buffer and then electrophoresed on a 12% polyacrylamide gels containing 7M
urea, followed by quantitation by direct radioanalytic imaging.
g. RNase Assays-Compound or sample was tested in various RNase assays as
follows. Radiolabeled ALMV substrate as in (f), was incubated at 5.5 nM
with 0.02 units/.mu.l of RNase T1 or 0.04 units/.mu.l of RNase U2 in 10 mM
NaCitrate, pH 5.0/0.5 mM EDTA/7M urea at 56.degree. C. for 20 min and
reaction products were electrophoresed on 12% polyacrylamide gels
containing 7M urea as in (f). RNase A at 4 ng/.mu.l was incubated with 5.5
nM radiolabeled ALMV substrate in 10 mM NaCitrate, pH 3.5/0.5 mM EDTA/7M
urea at 30.degree. C. for 20 min. and reaction products were
electrophoresed as above.
III. DATA FOR COMPOUND A
TABLE 3
______________________________________
BIOCHEMICAL SPECIFICITY OF COMPOUND A
______________________________________
Polymerase IC.sub.50 (.mu.M)
______________________________________
ALMV (880 nt)-primed flu transcription
1.1
ApG-primed flu transcription
>1000.0
VSV transcription >500.0
HIV Reverse Transcriptase
>300.0
T7 Phage RNA Polymerase
>100.0
Hela RNA Polymerase II >500.0
Hela DNA Polymerase-a >100.0
______________________________________
Nuclease IC.sub.50 (.mu.M)
______________________________________
HIV RNase H 100.0
U. sphaerogena RNase U2
>200.0
A. orzae RNase T1 >200.0
Bovine pancreatic RNase A
>200.0
EcoRI Restriction Endonuclease
>500.0
Influenza Endonuclease 1.8
______________________________________
Compound A was tested in biochemical assays at concentrations in the range
of 1.0-500 .mu.M as described in materials and methods.
TABLE 4
______________________________________
INHIBITION OF INFLUENZA A AND B VIRUS
TRANSCRIPTION WITH COMPOUND A
Virus Strain IC.sub.50 (.mu.M)
______________________________________
A/PR/8/34 (H1N1) 1.10
A/Japan/305/57 (H2N2)
0.25
A/Port Chalmers/1/73 (H3N2)
0.50
A/Hong Kong/8/68 (H3N2)
0.62
B/Hong Kong/5/72 0.85
______________________________________
Compound A was tested in in vitro transcription with various influenza
viruses primed with cap1 ALMV as described in material and methods.
While the foregoing specification teaches the principles of the present
invention, with examples provided for the purpose of illustration, it will
be understood that the practice of the invention encompasses all of the
usual variations, adaptations, or modifications, as come within the scope
of the following claims and its equivalents.
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